The on-site and sensitive detection of pathogens is of critical importance to the prevention, surveillance and control of infectious diseases and their outbreak at the first onset. While conventional techniques such as plate culturing, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) have been used as the predominant detection workhorses, they are limited by either time-consuming procedure, complicated sample pre-treatment, expensive analysis and operation, or inability to be implemented at point-of-testing. Significant efforts have been made to improve the limitations associated with conventional techniques. Among these, gold nanoparticles (AuNPs) have emerged as an excellent candidate for biosensor design owing to their unique properties. For example, colloidal AuNPs exhibit distinct colours and strong absorption bands in the visible range of the electromagnetic spectrum that are not present in the bulk metal. This fascinating optical phenomenon of AuNPs is derived from localized surface plasmon resonance (LSPR), a collective oscillation of free electrons in tandem with the incoming photon frequency. This has provided a range of simplified transducing mechanisms for biosensor design, based on assembly, disassembly, or enlargement of the AuNPs which allow scanometric, colorimetric or even naked-eye determination. Nucleic acid-modified AuNPs have been incorporated into biological sensing platforms to provide improved sensitivity, versatility and portability. Remarkably, the nucleic acid functionalized AuNPs not only provide further functionalities such as specific programmable assembly upon hybridization with their complementary counterparts, but also allow enzymatic cleavage, ligation and extension reactions for biosensor development.
Toward this end, studies have focused on incorporating nuclease enzymes and deoxyribozymes (DNAzyme) to cleave or link oligonucleotides to induce a colorimetric response. DNA endonuclease (DNase Pb2+-dependent RNA-cleaving DNAzyme (DNAzyme 8-17), exonuclease III (Exo III) and RNAse H have been used successfully for the detection of Pb2+, nucleic acids and folate receptor. DNAzyme 8-17, which cleaves the DNA substrate with a single RNA linkage in the presence of Pb2+, has been utilised for the detection of metal ions. In a different approach, incorporating the same 8-17 enzyme, cross-linking of enzyme-substrate and subsequent cleavage and dissociation of AuNPs upon the addition of target analyte (Pb2+) has been reported. Several studies have also focused on Exo III enzyme which catalyzes the stepwise removal of mononucleotides from blunt or recessed 3′-hydroxyl terminus of duplex DNA. A universal platform has been developed for the detection of DNA based on Exo III signal amplification. Furthermore, Exo III has been utilized for the colorimetric detection of folate receptor, in which the target induced AuNP aggregation. The utilization of Exo III enzyme has proven highly sensitive due to repeated hybridization and hydrolysis reactions. In a different approach, AuNPs were modified with EcoRI enzyme and a specific, double stranded DNA probe was designed which contained an EcoRI recognition site and complementary sticky ends. AuNP aggregation occurred in the presence of the target (magnesium and phosphate ions), resulting in a colorimetric response. Although highly successful, such enzymatic approaches are limited by the need for restriction binding sites, extensive probe design, and requirement for further amplification steps.
Herein, we present innovative sensing methods and kits based on the unique enzymatic activity of endonucleases, such as RNase H, for the detection of DNA, such as bacterial DNA, at concentrations down to femtomolar level. Due to the ubiquitous nature and high levels found in food, especially poultry, Campylobacter jejuni was chosen as the target for assay development and to exemplify the method of the invention (see Examples). The exemplified method utilizes RNA-functionalized AuNPs which form DNA-RNA heteroduplex structures through specific hybridization with target DNA. Once formed, the DNA-RNA heteroduplex is susceptible to RNase H enzymatic cleavage of the RNA probe, allowing DNA to liberate and hybridize with another RNA strand. This continuously happens until all, or substantially all, of the RNA strands are cleaved, leaving the nanoparticles unprotected, or substantially unprotected, and prone to aggregation upon exposure to a high electrolytic medium. The current invention overcomes previous limitations associated with enzyme-based methods in that it does not require further amplification steps. In addition, enzymes such as RNAse H which are not active on single stranded DNA or RNA molecules and only catalyze the cleavage of RNA within a DNA-RNA heteroduplex, do not require specific recognition sites for enzymatic cleavage. Furthermore, there is greater versatility and applicability with regard to probe design and thus potential for multiplexing. RNase H has previously been used for the detection of DNA via RNA cleavage within a DNA-RNA heteroduplex structure and subsequent release of a fluorescence dye to generate a fluorescence signal.[1] That method has a reported limit of detection (LOD) of 10 pM, which highlights the ultra-sensitivity of the method of the present invention which can detect target DNA at 1 pM as determined by the naked eye, or even down to femtomolar level by spectroscopic analysis (see Examples). The fluorescence-based approach mentioned above is further limited by cost due to the synthesis of fluorescein conjugate and requirement for equipment capable of detecting the fluorescence signals. The present invention is significantly different from previous reports as it utilizes the plasmonic properties of metal nanoparticles to produce a colorimetric response, in particular gold nanoparticles which produce a red-to-blue colorimetric response, thus the signal can be visibly detected by the naked eye. In addition, DNA detection can be performed at isothermal conditions in less than three hours. These advantages provide a basis for eradicating the need for a thermal cycler, complicated sample preparation, labelled fluorophores, and expensive and cumbersome read-out equipment. Finally, the application of the present invention to a food matrix has also been assessed and it is evident that the sensitivity and robustness of the assay is conducive for food safety analysis.